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Volume 93, Number 3, May-June 1988 Journal of Research of the National Bureau of Standards Accuracy in TraceAnalysis

Bioanalytical/

Biochemical Applicationsof chromatogram is moved in the x and y axes into Chromatography/SIMS and out of the point of instrument focus, defined as the locus of the primary beam, and the sec- ondary ion extraction optics of a quadrupole mass K. L. Busch, M. S. Stanley, K. L. Duffin, spectrometer. Data in four dimensions are gener- and J. C. Dunphy ated: x, y, m/z ratio, and relative abundances of the in the 12-4]. Department of Analytical advantages of the chromatography/ Indiana University SIMS instrument include an independent access or- Bloomington, IN 47405 der to sample spots, variable integration time for spectral measurement, variable spatial resolution The balanced capabilities of a high resolution adjusted in real time to maximize analysis effi- separation method coupled with a flexible detec- ciency, the ability to store sample as well as data tion method such as results generated in its analysis, preservation of all sample in powerful analytical methods including gas applied to the chromatogram, the ability to analyze chromatography/mass spectrometry and liquid chromatograms from outside sources, and the abil- chromatography/mass spectrometry. However, ity to calibrate the instrument with each chro- separations for diverse applications such as organic matogram [5]. We have demonstrated application synthesis, pharmacological analysis, and biochemi- of chromatography/SIMS to a variety of biochem- cal separations are still based on static chromatog- ical samples and have used the mass spectral data raphy, defined as a method in which the separation to identify compounds contained in the chro- of sample components is based on a spatial separa- matogram, and to increase the resolution of the tion, rather than on a difference in retention times. chromatographic separation. The mass spectrome- Although capabilities for separation of very com- ter is a "" that can either be operated as a plex mixtures for static chromatography (thin layer general detector, or in concert with functional- or or electrophoresis) do group-specific-derivatization reactions that have not rival the very high resolution achievable with been concurrently developed. the gas and liquid chromatography, the resolution Figure I illustrates part of the data array mea- is adequate for a large number of applications, and sured in an analysis of a mixture of chenodeoxy- the ease and low relative cost of the methods are cholic acid (A) and cholic acid (B) separated by advantageous. General detection methods for or- thin layer chromatography (metal-backed ganic compounds separated by these forms of chro- plate, 20 gm thickness). A dominant ion in the pos- matography still seem relatively crude. Color itive ion SIMS of chenodeoxycholic acid appears development, charring, staining, or fluorescence at m/z 357; similarly, the SIMS spectrum of cholic measurements provide the location of sample spots, acid contains an abundant ion at m/z 355. The mass but little information about sample identity. In spectral data acquired over the range of x and y many cases, sample identity is suggested on the ba- coordinates is reconstructed for ions of these sis of an R. identical with that of the standard. Such masses. These data were acquired with a cesium a match is a necessary but not a sufficient means of ion gun with moderate spatial resolution. The compound identification. maximum sample spot diameter is I mm, and 5 pag Over the past 2 years, we have constructed a of each compound were present in the spot. secondary ion mass spectrometer (SIMS) that ac- Spectra could be recorded for several hours in commodates large chromatograms within the each analysis (sample consumption rate is a few source, and sputters organic ions from the surfaces tens of picograms per second), and the same distri- to produce the mass spectrum of samples directly bution is recorded if the chromatogram is removed from the chromatogram. Details of instrument con- from the mass spectrometer, stored, and reanalyzed struction have been reported [1]. In essence, the later. 499 Volume 93, Number 3, May-June 1988 Journal of Research of the National Bureau of Standards Accuracy in Trace Analysis

Figure 2 represents the analysis of pyridostig- Vincze of the Israel Institute for Biological Re- mine bromide separated by thin layer chromatogra- search. phy. The structure of this drug is shown; the SIMS spectrum contains an abundant ion for the intact cation at m/z 223. Variation of this particular ion with x and y is shown in the abundance plot. The same data are shown plotted as isoabundance con- tours. Each ion in the SIMS spectrum derived from the sample exhibits similar contours, and pattern recognition programs can be used to link such ions together to form a mass spectrum of the compound free of background. The chromatography/SIMS method has been extended to the indirect analysis of electrophore- Figure 1. Reconstruction of the abundances of the ions at mAz tograms. Although aqueous gels can be dried for 357 (chenodeoxycholic acid, A) and mAz 355 (cholic acid, B) as a function of x and y coordinates of the thin layer chro- analysis, this often degrades the spatial resolution matogram on which they were separated. of the separation and makes the gel difficult to han- dle. The high vapor pressure of water (even at low PYRIDOSTIGMINEBROMIDE temperatures) precludes direct analysis of elec- TLC/SORBITOL I I trophoretic gels by SIMS. Standard sample trans- fer techniques such as Southern and Western blots w are used to transfer samples to nitrocellulose and diazotized media, then analyzed by SIMS. Bradykinin and a series of related have been so separated and analyzed; the SIMS spectra obtained are identical to those obtained from a dis- crete sample. _~~~~~~~.. Control of the physical and chemical nature of 0 the chromatographic surface underlies the success of chromatography/SIMS. For maximum sensitiv- Figure 2. Abundance and isoabundance contour plots of the ion to the intact cation of pyridostigmine ity, the sample should be in an ionic state, and at m/z 223 corresponding bromide, separated from related drugs in a silica gel thin layer should exhibit surfactant properties in the phase chromatogram. transition matrix used for the analysis [4]. Figure 3 is the positive ion SIMS spectrum of a mixture of RA900' 235 five pyrylium salts (10 jug each) used for derivatiza- tion of primary amine groups in peptides. The 80' 0 contains only ions from the recorded spectrum 70.01 pyrylium salt of the structure shown, which was synthesized specifically for its surface activity. No ions from the other pyrylium salts, or from the ma- trix, are observed. Several peptides have been 00.01 derivatized with this surface-active agent. In addi- tion to increased sensitivity, determination of the /'N sequence of the by MS/MS analysis of the 20 . 2 parent ion of the derivative is greatly simplified. 153 2'~i 1 60.01 l to. 2270. 250. 300 340. 310 Acknowledgments mlz Figure 3. Positive ion secondary ion mass spectrum of a mixture Our research in chromatography/SIMS has been of five pyrylium salts of equal , showing only the supported by the Whitaker Foundation, NIH, and ions corresponding to the pyrylium salt of the structure shown, NSF. The data on pyridostigmine bromide is illustrating the surfactant properties of this salt in the matrix derived from a collaborative study with Adam used.

500 Volume 93, Number 3, May-June 1988 Journal of Research of the National Bureau of Standards Accuracy in Trace Analysis

References peroxide, then reagents I and 2 would be luminol and catalyst. [1] Fiola, J. W., DiDonato, G. C., and Busch, K. L., Rev. Sci. Such a system requires of these Instrum. 57, 2294 (1986). reagents plus the necessary pumps, tubing, and [2] Busch, K. L., Fiola, J. W., DiDonato, G. C., Flurer, R. A., and Kroha, K. J., Adv. Mass Spectrom. 10, 855 (1986). mixers to deliver them and results in dilution and [3] DiDonato, G. C., and Busch, K. L., Anal. Chem. 58, 3231 broadening of the sample plug. Our research effort (1986). has explored various ways to contain these reagent [4] Stanley, M. S., and Busch, K. L., Anal. Chim. Acta 194, 199 components in solid-state or immobilized format so (1987). that the usual reagents solutions, pumping, and tub- [5] Busch, K. L., TrAC 6, 95 (1987). [6] Stanley, M. S., Duffin, K. L., Doherty, S. J., and Busch, K. ing can be eliminated, and the reagents incorpo- L., Anal. Chim. Acta, in press. rated in-line as indicated in figure 2. [7] Duffin, K. L., and Busch, K. L., manuscript in preparation. Immobilized luminol is of use for determinations of catalyst species or for determination of peroxide (and species such as glucose, cholesterol, etc., which can be enzymatically converted into an equivalent amount of peroxide). We have cova- lently bound luminol to silica, controlled-pore ChemiluminescenceDetection glass, and Ambersorb particles via various silane in Flowing Streams-Immobilized and linkage . By using glutaraldehyde to and Solid-State Reagents bridge between the amine group on luminol and the amine group on an aminoalkylsilane, loadings Timothy A. Nieman of 15 mg luminol/g support are achieved for sup- ports with about 400 square meters surface area/ Department of Chemistry gram. This material is packed into a column placed University of Illinois into the flow stream. The immobilized luminol is 1209 W. California St. stable in neutral , but injection of a portion Urbana, IL 61801 of an alkaline solution will release a controlled amount of luminol (via hydrolysis of the bond be- Chemiluminescence (CL) reactions yield light as tween luminol and glutaraldehyde); the amount re- one of their products. Luminol (5-amino-2,3-dihy- leased is dictated by the pH and volume of the dro-1,4-phthalazinedione) is one of the CL reagents solution injected. We have also contained luminol most commonly used in analysis. In aqueous alka- simply by onto Ambersorb and released line solution, luminol is oxidized to yield 3- it in the same manner. Advantages to release of aminophthalate and light. Perhaps the most luminol prior to the CL reaction include improve- analytically useful oxidant is hydrogen peroxide, ment in the CL quantum efficiency and increased for which a reaction catalyst is required. Typical flexibility in engineering the contact with the other catalysts are peroxidase, hemin, transition metal reagents. By use of this material in flow injection, a ions, or ferricyanide. The luminol CL system most of 0.1 M for peroxide is obtained. often used consists of luminol + H202 + catalyst An immobilized catalyst is of use for determina- + OH . Measurements of CL intensity can be used tion of peroxide (and analytes enzymatically con- to quantitate any of these species. Thus, luminol verted to peroxide) or analytes that have been CL has been used to determine catalysts or species labeled with luminol or related compounds. An ap- labelled with catalysts, peroxide, or species which proach that we have investigated involves use of can be converted into peroxide, and luminol, or electrogeneration of luminol CL. Luminol can be species labelled with luminol. oxidized at a positively-biased electrode, and CL For quantitative determination of any one spe- results if oxygen or hydrogen peroxide are present. cies involved in this reaction, one needs a way to Electrogeneration of luminol CL offers the unique deliver the other reaction components into the re- characteristics that the CL emission is confined to action zone or vessel. In flowing stream situations, near the electrode surface and that the reaction can either flow injection analysis or liquid chromatog- be turned "on" and "off' via control of electrode raphy, one would have a stream of analyte into potential. The net electrogenerated CL (ECL) in- which were flowed streams of the other necessary tensity due to peroxide injections appears as peaks reagents as shown in figure 1. If the analyte were on top of an appreciable and inescapable back-

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